JPS60258126A - Production of polymerized hemoglobin - Google Patents
Production of polymerized hemoglobinInfo
- Publication number
- JPS60258126A JPS60258126A JP59113525A JP11352584A JPS60258126A JP S60258126 A JPS60258126 A JP S60258126A JP 59113525 A JP59113525 A JP 59113525A JP 11352584 A JP11352584 A JP 11352584A JP S60258126 A JPS60258126 A JP S60258126A
- Authority
- JP
- Japan
- Prior art keywords
- hemoglobin
- erythrocytes
- blood
- glutaraldehyde
- resultant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
産業上の利用分野
この発明は、人工血液としての重合化ヘモグロビンの製
造方法に関するものである。DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application This invention relates to a method for producing polymerized hemoglobin as artificial blood.
従来技術
赤血球は、ヘモグロビンを高濃度に含んだ血液細胞であ
シ、ヘモグロビンは酸素結合能を有するヘムタンパク質
であるから、生体内における酸素運搬機能に重要な役割
を果たす。したがって、大意の出血などで生体内の赤血
球が減ると生命が危険に陥る。そこでこのような場合、
ＡＣＤ保存血の赤血球輸血が行なわれるのでおるか、血
清肝炎力どの副作用がしばしはみられる。Prior Art Red blood cells are blood cells containing a high concentration of hemoglobin, and since hemoglobin is a hemoprotein with oxygen binding ability, it plays an important role in the oxygen transport function in living bodies. Therefore, if the number of red blood cells in the body decreases due to bleeding, etc., life is at risk. So in such a case,
Because ACD-stored red blood cell transfusions are performed, side effects such as serum hepatitis and hepatitis are often observed.
一方、副作用を伴わない人工血液として、ヒトゝ赤血球
から得たヘモグロビンが考えられるが、その分子量はた
かだか約６万４千であるから、血管に住人するとすぐに
腎臓から尿へ排出されてしまい、所期の目的を満足に達
成することができない。On the other hand, hemoglobin obtained from human red blood cells can be considered as an artificial blood without side effects, but since its molecular weight is approximately 64,000 at most, it is immediately excreted from the kidneys into the urine after it resides in the blood vessels. Unable to satisfactorily achieve the intended purpose.
発明の目的
したがってこの発明の目的とするところは、輸血しても
副作用がなく、しかも長期間にわたシ廂液中に留り得る
人工血液を提供することにある。OBJECTS OF THE INVENTION Therefore, it is an object of the present invention to provide artificial blood that causes no side effects when transfused and that can remain in blood vessels for a long period of time.
問題点を解決するための手段
この発明の重合化ヘモグロビンの製造方法によると、赤
血球を低調処理して得たヘモグロビンにグルタルアルデ
ヒドを添加し重合せしめるのであ ：す、これを以下実
施例によシ詳しく説明する。Means for Solving the Problems According to the method for producing polymerized hemoglobin of the present invention, glutaraldehyde is added to hemoglobin obtained by low-temperature treatment of red blood cells and polymerized. explain in detail.
実施例
慢ず、ＡＣＤ保存血またはＣＰＤ保存血を冷凍高速遠心
分離機で遠心分陰（約８ｋｇ、＋分以内）し、血しょう
、顆粒球および赤血球に分陰する。EXAMPLE First, blood stored in ACD or blood stored in CPD is centrifuged (about 8 kg, within + minutes) using a frozen high-speed centrifuge to separate blood into plasma, granulocytes, and red blood cells.
得られたヒト赤血球に５倍以上容簾の０．９％塩化−ｔ
トリウム溶液を加ジる。そして、ふたたび遠心分離処理
（約８ｋｇ、１分以内）を施したのち、ヒト赤血球を洗
浄してとシ出す。洗浄後のヒト赤血球を低調処理すべく
約５倍容量のｌ０ｍＭ！Ｊン酸溶液（ＰＨ７，４）で溶
血し、ふたたび高速遠心分離処理（約１２に！ｉ＋、約
２０分）を施して赤血球膜成分を分−・除去する。この
ようにして得られた赤血球溶血液たるヘモグロビン１答
に０．６２５％グルタルアルテ゛ヒトを１０の１容添加
し、室温に約１時間放置する。ついで、この液をカラム
に通し、遊産したグルタルアルデヒドを分融除去すると
、目的とする重合化ヘモグロビンが得られる。0.9% chloride-t which is more than 5 times more concentrated in the obtained human red blood cells
Add thorium solution. After centrifuging again (approximately 8 kg, within 1 minute), the human red blood cells are washed and killed. Approximately 5 times the volume of 10mM for low-level processing of human red blood cells after washing! Hemolyze with J acid solution (PH7.4), and perform high-speed centrifugation again (approximately 12 to 1+, approximately 20 minutes) to remove red blood cell membrane components. To one volume of hemoglobin, which is the lysed red blood cell hemoglobin thus obtained, 1/10 volume of 0.625% glutaractin is added, and the mixture is left at room temperature for about 1 hour. Next, this liquid is passed through a column to dissolve and remove the produced glutaraldehyde, thereby obtaining the desired polymerized hemoglobin.
なお、前記カラムは、ろ適用樹脂を充填したガラス管か
らなり、前記樹脂としては、たとえばスエーデン国ファ
ルマシア社製のセファデツクスＧ−２５（商品名）を用
いることができる。The column is made of a glass tube filled with a filtration resin, and as the resin, for example, Sephadex G-25 (trade name) manufactured by Pharmacia, Sweden can be used.
前述のようにして得られた重合化ヘモグロビンを平板等
重点電気泳動法によシ分析したところ、第１図の（Ｃ）
に示すような電気泳動パターンが得られた。ただし、第
１図のｆａ）は重合化処理を施していないヘモグロビン
の電気泳動パターンを示し、（１）は０．８．３３％濃
度の、（ｄ）は０．５％濃度の各グルタルアルデヒドを
添加した場合の重合化ヘモグロビンの電気泳動パターン
をそれぞれ示している。When the polymerized hemoglobin obtained as described above was analyzed by plate focus electrophoresis, (C) in Figure 1 was obtained.
An electrophoretic pattern as shown in is obtained. However, fa) in Figure 1 shows the electrophoretic pattern of hemoglobin that has not been subjected to polymerization treatment, (1) shows the electrophoresis pattern of 0.8.33% concentration, and (d) shows the electrophoresis pattern of each glutaraldehyde at 0.5% concentration. The electrophoretic patterns of polymerized hemoglobin in the case of adding is shown in each case.
これよシ明らかなように、重合化処理前のヘモグロビン
がＰ）ｉ、７．．００等電点に単一バンドとして集束す
るのに対し、０．５〜０．８％濃度のグルタルアルデヒ
ドで重合化処理したヘモグロビンでは、Ｐ Ｈ６，’１
５以下の′等電点に多数のバンドと−して分布する。こ
れは、ヘモグロビンが修飾ヘモグロビンに変質している
ことを意味するのであるが、０゜８％を越えると第１図
の（（ｂ）にも与られるように異常が現われてくる。ま
た、添加するグルタルアルデヒドの濃度が０．５％未満
であると満足な反応が得られない。したがって、使用で
きるグルタルアルデヒドの濃度は領５〜０．８％、好ま
しくは０．６〜０．７％でおる。なお、濃度が１％を越
えるとヘモグロビンが部分的にゲル化をおこし、水に不
溶性となる。As is clear, hemoglobin before polymerization is P)i, 7. ．． 00 isoelectric point as a single band, whereas in hemoglobin polymerized with 0.5-0.8% concentration of glutaraldehyde, the P H6,'1
It is distributed as many bands at the isoelectric point of 5 or less. This means that hemoglobin has changed into modified hemoglobin, but when it exceeds 0.8%, abnormalities appear as shown in Figure 1 ((b). If the concentration of glutaraldehyde used is less than 0.5%, a satisfactory reaction cannot be obtained. Therefore, the concentration of glutaraldehyde that can be used is in the range of 5 to 0.8%, preferably 0.6 to 0.7%. Note that when the concentration exceeds 1%, hemoglobin partially gels and becomes insoluble in water.
かかる濃度のグルタルアルデヒドの最適添加量 ・１は
、ヘモグロビン１容に対し約：０分の１容（約９容量％
）であるが、前記濃度との関係において約７〜１５容量
％の範囲から選択できる。そして遊離したグルタルアル
デヒドは前述のようにカラムで除去されることになる。The optimum amount of glutaraldehyde added at such a concentration is approximately 1/0 volume (approximately 9% by volume) per volume of hemoglobin.
), but can be selected from a range of about 7 to 15% by volume in relation to the above concentration. The liberated glutaraldehyde will then be removed in the column as described above.
グルタルアルデヒドで重合化処理されたヘモグロビンの
分子量は、ＳＤＳ電気泳動法で分析したところ１２万以
上であった。一方、血液中に存在する血清タンパク質の
分子量は約７万以上であるから、この発明によって製造
された重合化ヘモグロビン全血管内に注入しても、腎臓
から尿へ排出されることはない。The molecular weight of hemoglobin polymerized with glutaraldehyde was 120,000 or more when analyzed by SDS electrophoresis. On the other hand, since the molecular weight of serum proteins present in the blood is approximately 70,000 or more, even if the polymerized hemoglobin produced according to the present invention is injected into all blood vessels, it will not be excreted from the kidneys into the urine.
また、前記重合化ヘモグロビンは、一般的なヘモグロビ
ンに比して数倍高い酸素親和性を示す。Furthermore, the polymerized hemoglobin exhibits an oxygen affinity several times higher than that of general hemoglobin.
これを第２図によシ説明すると、後者は特性曲線（ａ）
に示すように、５０％の酸素飽和度を与える酸この発明
は前述のように構成されるので、人工血液として輸血を
しても副作用がなく、長期間にわたって血液中に留まり
、しかも酸素親和性が高−いのみならず比較的簡便に製
造をすることができるという利点がある。To explain this with reference to Figure 2, the latter is the characteristic curve (a)
As shown in Figure 2, the present invention is constructed as described above, so even if it is transfused as artificial blood, there will be no side effects, it will remain in the blood for a long time, and it will have a high oxygen affinity. It has the advantage that it not only has a high yield, but also can be manufactured relatively easily.
第１図はヘモグロビンおよび重合化ヘモグロビンの各電
気泳動パターンを示す図、第２図はこの発明によシ得た
重合化ヘモグロビンおよび一般的なヘモグロビンの各酸
素分圧−酸素飽和度特性図である。
特許出願人 友田庫夫ほか２名FIG. 1 is a diagram showing the electrophoretic patterns of hemoglobin and polymerized hemoglobin, and FIG. 2 is a diagram showing the oxygen partial pressure-oxygen saturation characteristics of polymerized hemoglobin obtained according to the present invention and general hemoglobin. . Patent applicant Norio Tomoda and 2 others
Claims (1)
デヒドを添加し重合せしめることを特徴とする重合化ヘ
モグロビンの製造方法。A method for producing polymerized hemoglobin, which comprises adding glutaraldehyde to hemoglobin obtained by low-temperature processing of red blood cells and polymerizing it.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59113525A JPS60258126A (en) | 1984-06-02 | 1984-06-02 | Production of polymerized hemoglobin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP59113525A JPS60258126A (en) | 1984-06-02 | 1984-06-02 | Production of polymerized hemoglobin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPS60258126A true JPS60258126A (en) | 1985-12-20 |
Family
ID=14614548
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP59113525A Pending JPS60258126A (en) | 1984-06-02 | 1984-06-02 | Production of polymerized hemoglobin |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS60258126A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63297330A (en) * | 1987-05-05 | 1988-12-05 | ジェン−チャン シア | Pasteurizable and freeze driable hemoglobin-base substitute blood |
| JPH05502240A (en) * | 1990-08-17 | 1993-04-22 | バクスター、インターナショナル、インコーポレイテッド | Compositions based on hemoglobin oligomers and methods for their production |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS51109983A (en) * | 1975-02-27 | 1976-09-29 | Alza Corp |
-
1984
- 1984-06-02 JP JP59113525A patent/JPS60258126A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS51109983A (en) * | 1975-02-27 | 1976-09-29 | Alza Corp |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63297330A (en) * | 1987-05-05 | 1988-12-05 | ジェン−チャン シア | Pasteurizable and freeze driable hemoglobin-base substitute blood |
| JPH0532372B2 (en) * | 1987-05-05 | 1993-05-14 | Shia Jennchan | |
| JPH05502240A (en) * | 1990-08-17 | 1993-04-22 | バクスター、インターナショナル、インコーポレイテッド | Compositions based on hemoglobin oligomers and methods for their production |
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